The present invention relates to isolated nucleic acid molecules which encode human and murine proline dehydrogenase, and methods for determining susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder, bipolar disorder (BP), or major depressive disorder in a subject by determining levels of proline dehydrogenase (PRODH) in a bodily sample. Furthermore, the present invention comprises polymorphisms of the human proline dehydrogenase (PRODH) gene which correlate to a phenotype closely related to schizophrenia or a disease or disorder related thereto. The present invention also relates to various assays for drugs or agents that can treat schizophrenia or a disease or disorder related thereto.
It has been posited that the amino acid proline serves as a modulator of synaptic transmission in the mammalian brain, due to the selective expression of a brain specific high affinity proline transporter in a subset of glutamatergic pathways (Fremeau et al., 1996). Proline transporter is modulated by enckephalins, the expression of which may be decreased in the brains of patients with schizophrenia and elevated proline concentration. Furthermore, recent analysis indicates that endogenous extracellular proline may regulate the basal function of some glutamate synapses by maintaining them in a partially potentiated state. Also, elevated proline concentration has also been previously associated with behavioral and neurological effects.
Evidence of an association between schizophrenia susceptibility and hemizygous deletions in chromosome 22q11 has been reported. More specifically, three hemizygous cryptic deletions at 22q11 in a sample of 300 unrelated schizophrenic patients have been reported and characterized [Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7612 (1995); Karayiorgou et al., Amer. J. Med. Genet. 74, 677 (1997)]. The frequency of this microdeletion in the general population is estimated to be approximately 0.02% and no deletions were found in a sample of 200 healthy controls. The identified locus (approximately 1.5 Mb in size) is located in the proximal part of a region at chromosome 22q11 and has been implicated independently in schizophrenia susceptibility through linkage studies [Karayiorgou and Gogos, Neuron 19, 967-979 (1997)]. This locus overlaps with the critical region involved in the etiology of Velocardio-facial (VCFS)/DiGeorge (DGS) syndromes [Driscoll et al. 1993]. Furthermore, it has been shown that approximately 29% VCFS children with 22q11 deletions develop schizophrenia or schizoaffective disorder in adolescence and adulthood [Pulver et al., 1994], an estimate confirmed by a more recent independent study [Murphy and Owen, Am. J. Med. Genet., 74, 660 (1997)]. Deletions in chromosome 22, band q11 (22q11) have been identified among schizophrenia patients of diverse ethnic origins (Chinese, Israeli, British, Danish [L. Y. Chow et al., Am. J. Med. Genet. 74, 677 (1997); D. Gothelf et al., Am. J. Med. Genet. 72, 455 (1997); O. Mors and H. Ewald, Am. J. Med. Genet. 74, 677 (1997); Hodginson et al, Am. J. Med. Genet. 61, 565 (1997)]) and the 22q11 region has been implicated in early-onset schizophrenia [Yan et al., 1998]. In addition, the increased rates of comorbid obsessive compulsive disorder (OCD) or symptoms (OCS) among schizophrenic patients with the 22q11 microdeletion locus [Karayiorgou et al., 1996, 1997; Papolos et al., 1996] and similarly increased rates of anxiety, OCS and OCD in children and adults with the 22q11 microdeletion in the absence of schizophrenia [Papolos et al., 1996], potentially indicate that the 22q11 genomic region may harbor one or more genes predisposing to obsessive compulsive disorder (OCD).
Moreover, it has been observed that approximately 20% of schizophrenia patients report obsessions and compulsions, features that are found in only 1-2% of the general population [Eisen and Rasmussen 1993; Berman et al., 1995]. Hence, it is possible that schizophrenia and OCD may share some pathophysiological and genetic components. One common central processing mechanism that seems to be affected in patients with schizophrenia and OCD is sensorimotor gating. Patients with schizophrenia and OCD demonstrate poor sensorimotor gating of the startle response as measured by impaired prepulse inhibition of an acoustic response, and this may lead to sensory overload, distractibility and cognitive fragmentation.
However, there is no genetic marker available which is indicative of a subject""s susceptibility to schizophrenia, or a disease related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP), or major depressive disorder (MDD).
Accordingly, what is needed is a genetic marker to assess a subject""s susceptibility to schizophrenia or a disease or disorder related thereto. Also needed is a genetic marker to diagnose schizophrenia, and the development of potential drugs or agents that have applications in treating schizophrenia or a disease or disorder related thereto, such as OCD, bipolar disorder, or major depressive disorder.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
There is provided, in accordance with the present invention, an isolated nucleic acid molecule which encodes human proline dehydrogenase, and the amino acid sequence of human proline dehydrogenase. Also provided is an isolated nucleic acid molecule comprising a DNA sequence which encodes murine proline dehydrogenase, and the amino acid sequence of murine proline dehydrogenase. Furthermore, there is provided, in accordance with the present invention, methods for determining a subject""s susceptibility to schizophrenia or a disease or disorder related thereto, such as a schizoaffective disorder or disorders related thereto, like OCD, bipolar disorder, or major depressive disorder, using a variant allele of the gene encoding PRODH. Detection of such a variant allele in the genome of a subject may be indicative of the subject""s susceptibility to schizophrenia. Furthermore, a variant allele of the PRODH gene can also be used to assay drugs and agents for potential use in treating schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder (MDD).
Thus broadly, the present invention extends to an isolated nucleic acid molecule encoding human proline dehydrogenase, wherein the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Furthermore, the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to the isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
The present invention also extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridzable thereto under standard hybridization conditions, wherein the nucleic acid molecule is detectably labeled. Numerous detectable labels have applications in the present invention. Examples include a radioactive element, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re, to name only a few, chemicals which fluoresce, or enzymes such as alkaline phosphatase or horseradish peroxidase conjugated to an isolated nucleic acid molecule of the invention.
Moreover, the present invention extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable under standard hybridization conditions thereto, wherein the nucleic acid molecule encodes human PRODH comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
Naturally, the present invention extends to an isolated human proline dehydrogenase protein (PRODH) comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
Also, the present invention extends to an antibody having human proline dehydrogenase comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof, as an immunogen. Such an antibody can be polyclonal, monoclonal, or chimeric. Further, an antibody of the invention having human PRODH as an immunogen can be detectably labeled. As explained above, examples of detectable labels having applications herein include, but certainly are not limited to radioactive isotopes, such as 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re, to name only a few. Chemicals which fluoresce, or enzymes such as alkaline phosphatase or horseradish peroxidase, can also be used as detectable labels.
In addition, the present invention extends to cloning vectors for creating copies or xe2x80x9ccloningxe2x80x9d an isolated nucleic acid molecule of the invention. More specifically, the present invention extends to a cloning vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, and an origin of replication. In another embodiment, the invention extends to a cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous cloning vectors which are commercially available to the skilled artisan can be used as a cloning vector of the invention. Further, it is readily within the skill of one of ordinary skill in the art to insert an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions into a readily available cloning vector using recombinant DNA techniques to produce a cloning vector of the invention. In particular, numerous commercially available cloning vectors have a polylinker site. One of ordinary skill in the art can readily cut open a cloning vector at its polylinker using a variety restriction endonucleases, and then insert the isolated nucleic acid molecule into the vector using DNA ligase. Furthermore, a skilled artisan can also manipulate the ends of an isolated nucleic acid molecule of the invention or fragment thereof to comprise particular restriction sites, and cut those cites with restriction endonucleases which also were used to cut open the vector. The restricted isolated nucleic acid molecule of the invention can then be readily inserted into a cloning vector of the invention. Any remaining gaps in the DNA sequence of the vector can then be filled in using individual deoxynucleotides and DNA ligase. Particular cloning vectors which have applications in the present invention include, but are not limited to E. coli, bacteriophages such as lambda derivatives, plasmids such as pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectors, or pmal-c, pFLAG, to name only a few.
Naturally, the present invention extends to an expression vector for expressing an isolated nucleic acid molecule of the invention in order to produce human PRODH, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In particular, an expression vector of the invention comprises an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. In another embodiment, an expression vector of the invention comprises an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridzable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous expression vectors can be used to express an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions. In particular, such expression vectors are generally commercially available to the skilled artisan, and like cloning vectors, comprise polylinker sites. As a result, commercially available expression vectors can be manipulated in a fashion similar to to the manipulation of a cloning vector, which is described above. Hence a skilled artisan can readily insert an isolated nucleic acid molecule of the invention into an expression vector such that the isolated nucleic acid molecule is operatively associated with a promoter. Examples of expression vectors having applications herein are described infra.
Moreover, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule encodes a human proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. In another embodiment, the invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule comprises a DNA sequence hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof. Numerous unicellular hosts which are readily available to the skilled artisan have applications in the present invention. Examples include, but certainly are not limited to, E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.
Naturally, the present invention extends to method for producing a PRODH comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, the method comprises the steps of:
a) culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule which comprises a DNA sequence of SEQ ID NO:1, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule to produce a protein comprising an amino acid sequence of SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the method comprises the steps of:
a) culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule, to produce a protein comprising an amino acid sequence of SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or an analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the present invention extends to an isolated nucleic acid molecule which encodes a human PRODH, wherein the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Furthermore, the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to the isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
The present invention also extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridzable thereto under standard hybridization conditions, wherein the nucleic acid molecule is detectably labeled. Numerous detectable labels have applications in the present invention and are described infra.
Moreover, the present invention extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable under standard hybridization conditions thereto, which encodes human PRODH comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
In addition, the present invention extends to cloning vectors for creating copies or xe2x80x9ccloningxe2x80x9d an isolated nucleic acid molecule of the invention. More specifically, the present invention extends to a cloning vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, and an origin of replication. In another embodiment, the invention extends to a cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous cloning vectors which are commercially available to the skilled artisan can be used as a cloning vector of the invention. Further, it is readily within the skill of one of ordinary skill in the art to insert an isolated nucleic acid molecule of the present invention or fragment thereof into a readily available cloning vector using recombinant DNA techniques to produce a cloning vector of the invention. What""s more numerous cloning vectors having applications herein are readily available to the skilled artisan, such that with the use of routine experimental techniques and the new and useful isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, one of ordinary skill can readily produce a cloning vector of the invention. Naturally, routine recombinant DNA techniques describe here can apply to the insertion of any isolated nucleic acid molecule of the invention in to a cloning vector.
Also, the present invention extends to an expression vector for expressing an isolated nucleic acid molecule of the invention in order to produce human PRODH, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In particular, an expression vector of the invention comprises an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. In another embodiment, an expression vector of the invention comprises an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridzable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous expression vectors can be used to express an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions. In particular, such expression vectors are generally commercially available to the skilled artisan, and like cloning vectors, comprise polylinker sites. As a result, commercially available expression vectors can be manipulated in a similar fashion in which cloning vectors of the invention are manipulated. Hence a skilled artisan can readily insert an isolated nucleic acid molecule of the invention into an expression vector such that the isolated nucleic acid molecule is operatively associated with a promoter. Examples of expression vectors having applications herein are described infra.
Moreover, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule encodes a human proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. In another embodiment, the invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule comprises a DNA sequence hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof. Numerous unicellular hosts which are readily available to the skilled artisan have applications in the present invention, and examples are described above.
Naturally, the present invention extends to method for producing a PRODH comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, a method for producing human proline dehydrogenase comprises the steps of:
c) culturing a unicellular host of transformed or transfected with an expression vector comprising an isolated nucleic acid molecule which comprises a DNA sequence of SEQ ID NO:9, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule to produce a protein comprising an amino acid sequence of SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or analog or derivative thereof; and
d) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the method comprises the steps of:
a) culturing a unicellular host of transformed or transfected with an expression vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule, to produce a protein comprising an amino acid sequence of SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or an analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the present invention extends to an isolated nucleic acid molecule which encodes murine proline dehydrogenase protein (Prodh). In particular, the present invention extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. Naturally, the present invention extends to an isolated nucleic acid molecule which is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. As explained above, isolated nucleic acid molecules of the invention can be detectably labeled. Examples of detectable labels having applications herein are described infra.
In addition, the present invention extends to an isolated nucleic acid molecule which encodes a Prodh protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. A particular example of such an isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Naturally, the present invention extends to an isolated Prodh protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
Further, the present invention extends to an antibody having a murine proline dehydrogenase protein, a fragment thereof, a conservative variant thereof, or an analog or derivative thereof as an immunogen. In a particular example, the immunogen of an antibody of the invention is a protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. Furthermore, such an antibody can be polyclonal, monoclonal or chimeric, and optionally can be detectably labeled.
The present invention further extends to cloning vectors which can replicate or xe2x80x9cclonexe2x80x9d an isolated nucleic acid molecule which encodes a murine proline dehydrogenase protein, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, a cloning vector of the invention comprises an origin of replication and an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. Another embodiment of a cloning vector of the invention comprises an isolated nucleic acid molecule and an origin of replication, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. As explained above, numerous commercially available cloning vectors which comprise a polylinker region have ready applications in the present invention. One of ordinary skill in the art can readily insert an isolated nucleic acid molecule of the invention into a commercially available cloning vector using routine recombinant DNA techniques, described infra. Particular examples of cloning vectors having applications herein include, but certainly are not limited to E. coli, bacteriophages, plasmids, or pUC plasmid derivatives. Furthermore, bacteriophage vectors having applications herein include lambda derivatives, plasmids further comprise pBR322 derivatives, and pUC plasmid derivatives further comprise pGEX vectors, or pmal-c, pFLAG, to name only a few.
In addition, the present invention extends to expression vectors for producing a murine proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In particular, the invention extends to an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. In another embodiment, an expression vector of the invention comprises an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridizable conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. Numerous expression vectors that are commercially available have applications herein. Examples of readily available vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage xcex, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2xcexc plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. Insertion of an isolated nucleic acid molecule into such an expression vector is readily within the skill of one of ordinary skill in the art using recombinant DNA techniques described herein.
Further, numerous promoters have applications herein. Examples of such promoters include, but certainly are not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac system the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast xcex1 mating factor, to name only a few.
The present invention further extends to unicellular hosts transformed or transfected with an expression vector of the invention. In particular, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. In another embodiment, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivative thereof. Examples of unicellular hosts having applications herein include but certainly are not limited to E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
Naturally, the present invention extends to methods of producing a murine proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, the method comprises the steps of:
a) culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule to produce a protein comprising an amino acid sequence of SEQ ID NO:4, conservative variant thereof, fragment thereof, or analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the method comprises the steps of:
a) culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof under conditions that provide for expression of the isolated nucleic acid molecule to produce a protein comprising an amino acid sequence of SEQ ID NO:4, conservative variant thereof, fragment thereof, or analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
The present invention further extends to an isolated variant allele of a human proline dehydrogenase (PRODH) gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human PRODH gene, wherein the isolated variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, and the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
In addition, the present invention extends to a detectably labeled isolated variant allele of a PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Numerous detectable labels have applications in the present invention. For example the detectable label can be a radioactive element, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re, to name only a few. Chemicals which fluoresce, or enzymes such as alkaline phosphatase or horseradish peroxidase, can also be used as detectable labels.
Moreover, the present invention extends to a detectably labeled isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Detectable labels set forth throughout the specification have applications in such an isolated nucleic acid molecule.
In addition, the present invention extends to an isolated variant allele of a PRODH gene which encodes a variant human proline PRODH comprising at least one variation in the amino acid sequence of PRODH, wherein PRODH comprises an amino acid sequence of SEQ ID NO:2, and is encoded by a human proline dehydrogenase gene comprising a DNA sequence of SEQ ID NO:1. A variant PRODH protein of the present invention comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the at least one variation comprises:
Arg101Trp;
Arg101Glu;
Glu437Arg; or
a combination thereof.
In particular, a variant allele of a human PRODH gene comprising a DNA sequence comprising a C to T transition in the first position of codon 101 of SEQ ID NO:1, encodes a variant human PRODH protein comprising an amino acid sequence comprising an Arg101Trp variation in SEQ ID NO:2. Furthermore, a variant allele of a human PRODH gene comprising a DNA sequence having a G to A transition in the second position of codon 101 of SEQ ID NO:1 encodes a variant human PRODH protein comprising an amino acid sequence having an Arg101Glu variation in SEQ ID NO:2. Hence naturally, a variant allele of a human PRODH gene comprising a DNA sequence having an A to G transition in the second position of codon 437 of SEQ ID NO:1 encodes a variant human PRODH protein comprising an amino acid sequence having a Glu437Arg variation in SEQ ID NO:2. As explained above, the present invention also extends to a variant allele of a human PRODH gene comprising a combination of variations set forth herein, which encode a variant human PRODH protein comprising an amino acid sequence having a combination of amino acid residue variations in SEQ ID NO:2 as described above.
Naturally, the present invention extends to an isolated variant human PRODH protein comprising an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the at least one variation comprises:
Arg101Trp;
Arg101Glu;
Glu437Arg; or
a combination thereof.
Furthermore, the present invention extends to an antibody having a variant proline dehydrogenase protein of the present invention as an immunogen. Such an antibody can be a polyclonal antibody, a monoclonal antibody, or a chimeric antibody. Moreover, an antibody of the present invention can be detectably labeled. Examples of detectable labels which have applications in this embodiment comprises a radioactive element, a chemical which fluoresces, or an enzyme, to name only a few.
In addition, the present invention extends to cloning vectors that can be used to clone copies of a variant allele of a PRODH gene of the present invention. An example of such a cloning vector comprises an origin of replication and an isolated variant allele of a human PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and an isolated variant allele of the PRODH gene comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Moreover, the present invention extends to a cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the isolated variant allele of the PRODH gene comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Numerous cloning vectors have applications in the present invention and are readily available to a skilled artisan. Furthermore, it is well within the knowledge of one of ordinary skill in the art to insert an isolated PRODH variant allele of the present invention into a commercially available cloning vector using recombinant DNA techniques described infra. Examples of a cloning vector having applications in the present invention include E. coli, bacteriophages, such as lambda derivatives, plasmids, such as pBR322 derivatives, and pUC plasmid derivatives, such as pGEX vectors, or pmal-c, pFLAG, to name only a few.
The present invention further extends to a unicellular host transformed or transfected with a cloning vector which comprises an isolated variant allele of the human PRODH gene as described above. Examples of hosts which are readily available to the skilled artisan and can be transformed or transfected with a cloning vector of the present invention include, but are not limited to E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
The present invention further extends to a method of cloning or producing xe2x80x9ccopiesxe2x80x9d of an isolated variant allele of a human PRODH gene of the invention, which comprises inserting a cloning vector into a unicellular host, and then inducing the host to self replicate. During the self replication of the host, the origin of replication of the cloning vector causes the replication of the cloning vector. After the unicellular host has self replicated numerous times, the cloning vectors can be isolated from the cloned host, and the isolated nucleic acid molecule can be isolated via restriction digestion from the cloning vectors.
Naturally, the present invention extends to expression vectors comprising an isolated variant allele of a PRODH gene operatively associated with a promoter, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and an isolated variant allele of the present invention comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Furthermore, the present invention extends to an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and an isolated variant allele of the present invention comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof.
Numerous promoters which are readily available to a skilled artisan, have applications in any expression vector of the invention. For example, immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast a mating factor, to name only a few, have applications herein. Furthermore, it is well within the knowledge of one of ordinary skill in the art to insert an isolated variant allele of the invention, or an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the invention into a commercially available expression vector, using recombinant DNA techniques described infra.
In addition, the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention. Examples of hosts which can be transformed or transfected with an expression vector of the present invention, and have applications in the present invention, include, but are not limited to, E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
Naturally, the present invention extends to a method for producing a variant human PRODH protein comprising an amino acid having at least one variation in SEQ ID NO:2, wherein the at least one variation comprises
Arg101Trp;
Arg101Glu;
Glu437Arg; or
a combination thereof.
An example of such a method comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele of a PRODH gene operatively associated with a promoter, wherein the isolated variant allele of the PRODH gene comprises a DNA sequence having at least one variation in SEQ ID NO:1, and the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof,
under conditions that provide for expression of the variant allele. The variant PRODH protein produced from such expression is then recovered from the unicellular host, the culture, or both.
Yet another method of the present invention for producing a variant PRODH protein involves culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a PRODH gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises:
a G to A transition in the third position of codon 83;
a C to T transition in the first position of codon 101;
a G to A transition in the second position of codon 101;
a C to T transition in the first position of codon 247;
a C to T transition in the third position of codon 342;
a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437;
a T to C transition in the first position of codon 497; or
a combination thereof,
under conditions that provide for expression of the isolated nucleic acid molecule. The variant human PRODH protein produced from such induced expression is then recovered from the unicellular host, the culture, or both.
Furthermore, the present invention extends to a method for detecting a susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder in a subject, wherein the method comprises measurement of the levels of activity of an enzyme in a bodily sample which is involved in proline catabolism. A comparison of the measurement of the levels of activity of the enzyme in the bodily sample is then made with the levels of activity of the enzyme in a standard. A modulated level of enzyme activity in the sample relative to the level of activity in the standard is indicative of a susceptibility to, or the presence of, schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder, in the subject. In a particular embodiment, the enzyme involved in proline catabolism is proline dehydrogenase (PRODH), and a reduced level of activity of PRODH in a bodily sample from the subject compared to the level of PRODH activity in the standard is indicative of increased susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto in the subject relative to the susceptibility of the standard. Methods of assaying activity of proline dehydrogenase in a bodily sample are readily available to the skilled artisan.
The present invention further extends to a method for determining a susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto in a subject, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder (MDD), wherein the method comprises the steps of:
a) removing a bodily sample from the subject, wherein the sample comprises a PRODH gene; and
b) determining whether the PRODH gene of the bodily sample comprises a DNA sequence having a variation in SEQ ID NO:1 comprising a T to C transition in the first position of codon 497. The presence of the variant allele the PRODH gene in a bodily sample of the subject indicates the subject has an increased susceptibility to schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder (MDD) relative to the susceptibility of a standard, wherein the bodily sample of the standard comprises a PRODH gene comprising a DNA sequence of SEQ ID NO:1.
Furthermore, the present invention extends to an assay for screening drugs and other agents for ability to treat schizophrenia or a disease or disorder related thereto. Such an assay of the present invention comprises the steps of culturing an observable cellular test colony which produces PRODH and which has been inoculated with the drug or agent to be assayed, harvesting a cellular extract from the cellular test colony, and determining the level of activity of PRODH in the test colony. An increase or decrease in the level of activity of PRODH in this test colony compared to a control test colony not inoculated with the drug, or compared to the level of activity of PRODH in the cellular test colony prior to inoculation with the drug or agent, is indicative of the ability of the drug or agent to modulate the production, stability, degradation or activity of PRODH, which in turn is indicative of the drug or agent""s ability to treat schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder. An increase in the level of activity of PRODH in the test colony after inoculation with the drug or agent compared to the level of activity in the control colony, or in the cellular test colony prior to inoculation with the drug or agent, indicates the drug has the ability to be used to treat schizophrenia or a disorder related thereto.
In another embodiment, the present invention extends to an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence and/or activity of PRODH, or to identify drugs or other agents that may potentiate or increase such activity. Broadly, a system or test kit of the present invention may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling the label to PRODH, its agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined, or their binding partner(s). The system or test kit may also comprise a polymerase chain reaction based (PCR) assay which can be used to quantify the PRODH levels of a sample.
Hence, the present invention extends to a test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder, comprising:
(a) a predetermined amount of a detectably labeled specific binding partner of a PRODH protein;
(b) other reagents; and
(c) directions for use of said kit.
Examples of the labeled immunochemically reactive component of such a test kit can be selected from the group consisting of polyclonal antibodies to PRODH, monoclonal antibodies to PRODH, chimeric antibodies to PRODH, fragments of such antibodies, and mixtures of such antibodies.
Furthermore, the present invention extends to a test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto in a subject, wherein the test kit comprises:
(a) PCR oligonucleotide primers suitable to detecting a variant allele of the PRODH gene in a sample;
(b) other reagents; and
(c) directions for use of the kit.
The present invention further extends to a test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto in a eukaryotic cellular sample, wherein the test kit comprises:
(a) PCR oligonucleotide primers suitable for detection of an isolated variant allele of a PRODH gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the isolated variant comprises a DNA sequence comprising a T to C transition in the first position of codon 497 of SEQ ID NO:1;
(b) other reagents; and
(c) directions for use of the kit.
In another embodiment, the present invention extends to treating schizophrenia or a disease or disease or disorder related thereto in a subject, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder. An example of such a method comprises administering to the subject a therapeutically effective amount of a composition comprising PRODH, wherein PRODH comprises an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. Optionally, the composition of the invention can be administered alone or in combination with additional therapeutic agents to treat the subject.
In addition, the present invention extends to a method for determining the schizophrenic-related pharmacological activity of an agent, wherein the method comprises the steps of:
administering the agent to a mammal;
determining the level of activity of PRODH in the mammal; and
comparing the level of activity of PRODH in the mammal to the level of activity of PRODH in a control mammal to which the agent was not administered. An increase in the level of activity of PRODH in the mammal relative to activity of PRODH in the control mammal indicates the agent has a schizophrenic-related pharmacological activity, and potential as a therapeutic agent for treating schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder.
In yet another aspect, the present invention extends to a method for determining the schizophrenic-related pharmacological activity of an agent, wherein the method comprises the steps of:
determining a basal level of activity of PRODH in the mammal;
administering the agent to the mammal;
determining the level of activity of PRODH in the mammal after administration of the agent; and
comparing the level of activity of PRODH after administration of the agent to the basal level of activity.
An increase in the level of activity of PRODH in the mammal relative to the basal level in the mammal indicates the compound has a schizophrenic-related pharmacological activity, and may have potential as a therapeutic agent for treating schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder.
What""s more, the present invention extends to an isolated variant allele of the Prodh gene which encodes a mutated murine Prodh protein. In particular, the present invention extends to an isolated variant allele of the Prodh gene, wherein the isolated variant allele comprises a DNA sequence of FIG. 10 (SEQ ID NO:7), degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
The present invention also extends to an isolated nucleic acid molecule hybridzable under standard hybridization conditions to the isolated variant allele of the murine Prodh gene comprising a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
In addition, the present invention extends to an isolated variant allele of the Prodh gene which comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, detectably labeled. Naturally, the present invention extends to an isolated nucleic acid molecule detectably labeled, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of the Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule encoding an isolated mutant murine Prodh protein comprising an amino acid sequence of FIG. 11 (SEQ ID NO:8), conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
Naturally, the present invention extends to an isolated Prodh comprising an amino acid sequence of SEQ ID NO:8, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.
Also, the present invention extends to an antibody having an isolated mutant Prodh of the invention, conservative variants thereof, fragments thereof, or analogs or derivatives thereof as an immunogen. Such an antibody can be polyclonal, monoclonal, or chimeric. Further, such an antibody can be detectably labeled. As explained above, numerous examples of detectable labels having applications in an antibody of the invention are described infra.
In addition, the present invention extends to cloning vectors for creating copies or xe2x80x9ccloningxe2x80x9d an isolated variant allele of a Prodh gene of the invention, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. In particular, the present invention extends to a cloning vector comprising an isolated variant allele of a Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, and an origin of replication. In another embodiment, the invention extends to a cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous cloning vectors which are commercially available to the skilled artisan, and can be used as a cloning vector for an isolated variant allele of a Prodh gene. Examples of such cloning vectors, and routine recombinant DNA techniques to produce such a vector are described infra.
Naturally, the present invention extends to an expression vector for expressing an isolated variant allele of a murine Prodh gene, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, along with an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions, to produce a mutated murine Prodh protein, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In particular, an expression vector of the invention comprises an isolated variant allele of the murine Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. In another embodiment, an expression vector of the invention comprises an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridzable under standard hybridization conditions to an isolated variant allele of the Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Numerous expression vectors can be used to express the isolated variant allele of the Prodh gene, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable to the isolated variant allele under standard hybridization conditions. In particular, such expression vectors are generally commercially available to the skilled artisan, and like cloning vectors, comprise polylinker sites. As a result, commercially available expression vectors can be manipulated in a similar fashion in which cloning vectors of the invention are manipulated. Hence a skilled artisan can readily insert the isolated variant allele of the Prodh gene, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions into an expression vector such that the isolated variant allele or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions is operatively associated with a promoter. Examples of expression vectors having applications herein are described infra.
Moreover, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele of the Prodh gene, operatively associated with a promoter, wherein the isolated variant allele encodes a mutant murine proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:8, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, the present invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele of the Prodh gene, operatively associated with a promoter, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. In another embodiment, the invention extends to a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of the Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof. Numerous unicellular hosts which are readily available to the skilled artisan have applications in the present invention.
Naturally, the present invention extends to method for producing a mutant murine proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:8, conservative variants thereof, fragments thereof, or analogs or derivatives thereof. In one embodiment, a method for producing a mutant murine proline dehydrogenase comprises the steps of:
a) culturing a unicellular host of transformed or transfected with an expression vector comprising an isolated variant allele of the prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated variant allele to produce a mutant murine proline dehydrogenase protein comprising an amino acid sequence of SEQ ID NO:8, a conservative variant thereof, a fragment thereof, or analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
In another embodiment, the method comprises the steps of:
a) culturing a unicellular host of transformed or transfected with an expression vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the Prodh gene, wherein the isolated variant allele comprises a DNA sequence of SEQ ID NO:7, a degenerate variant thereof, a fragment thereof, or an analog or derivative thereof, operatively associated with a promoter, under conditions that provide for expression of the isolated nucleic acid molecule, to produce a protein comprising an amino acid sequence of SEQ ID NO:8, a conservative variant thereof, a fragment thereof, or an analog or derivative thereof; and
b) recovering the protein from the unicellular host, the culture, or both.
Furthermore, the present invention extends to a method for identifying a drug or agent for treating schizophrenia or a disease or disorder related thereto. An example of such a method comprises the steps of:
performing an first pre-pulse inhibition test (PPI) test on a mouse having within its genome two copies of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein both copies are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8, to obtain a first percentage of inhibition of startle response;
administering the potential drug or agent to the mouse;
performing a second PPI test on the mouse to obtain a second percentage of inhibition of startle response; and
comparing the first percentage to the inhibition of startle response with the second percentage of startle response,
wherein an increase in percentage of inhibition in the second percentage of inhibition relative to the first percentage of inhibition is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto. Thus, if the percentage of inhibition of startle response in the mouse having within its two active copies of an isolated variant allele of a Prodh gene comp mouse after administration of the drug or agent is greater than the percentage of inhibition of startle response in the Pro/Re mouse prior to inhibition, then the drug or agent has the ability to treat schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder.
Also, the present invention extends to a method for identifying a drug or agent for treating schizophrenia or a disease or disorder related thereto. An example of such a method comprises the steps of:
performing an first pre-pulse inhibition test (PPI) test on an F3 generation mouse from a cross Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8, to obtain a first percentage of inhibition of startle response;
administering the potential drug or agent to the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type;
performing a second PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type to obtain a second percentage of inhibition of startle response; and
comparing the first percentage to the inhibition of startle response with the second percentage of startle response,
wherein an increase in percentage of inhibition in the second percentage of inhibition relative to the first percentage of inhibition is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto.
What""s more, the present invention extends to a method for identifying a drug or agent for use in treating schizophrenia or a disease or disorder related thereto, comprising the steps of:
a) administering the drug or agent to an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;
b) performing a PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type to obtain a percentage of inhibition of the startle response in the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type which was administered the drug or agent; and
c) comparing the percentage of inhibition of the startle response in the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type with the percentage of inhibition of the startle response in an unmedicated F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8.
An increase in percentage of inhibition in the percentage of inhibition in the medicated mouse relative to the percentage of inhibition in the unmedicated mouse is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto.
Furthermore, the present invention extends to a method for identifying a drug or agent for use in treating schizophrenia or a disease or disorder related thereto, comprising the steps of:
a) administering the drug or agent to a mouse having within its genome two copies of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein both copies are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;
b) performing a PPI test on the mouse to obtain a percentage of inhibition of the startle response in the mouse; and
c) comparing the percentage of inhibition of the startle response in the mouse with the percentage of inhibition of the startle response in an unmedicated mouse having within its genome two copies of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein both copies are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8.
An increase in percentage of inhibition in the percentage of inhibition in the medicated mouse relative to the percentage of inhibition in the unmedicated mouse is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto.
In addition, the present invention extends to a method for identifying a drug or agent for use in treating schizophrenia or a disease or disorder related thereto, comprising the steps of:
a) administering the drug or agent to a mouse having within its genome two copies of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7, wherein both copies are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;
b) performing a PPI test on the mouse to obtain a percentage of inhibition of the startle response in the mouse; and
c) comparing the percentage of inhibition of the startle response in the mouse with the percentage of inhibition of the startle response in an unmedicated mouse having within its genome two copies of an isolated Prodh gene comprising a DNA sequence of SEQ ID NO:3, wherein both copies are capable of expressing a Prodh comprising an amino acid sequence of SEQ ID NO:4.
If the percentage of inhibition of the startle response in the medicated mouse is statistically equivalent to the percentage of inhibition in the mouse capable of expressing Prodh comprising a DNA sequence of SEQ ID NO:4, then the drug or agent has the ability to treat schizophrenia or a disease or disorder related thereto.
In another embodiment, the present invention extends to an a method for identifying a drug or agent for use in treating schizophrenia or a disease or disorder related thereto, comprising the steps of:
a) administering the drug or agent to an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;
b) performing a PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type administered the drug or agent to obtain a percentage of inhibition of the startle response in the mouse; and
c) comparing the percentage of inhibition of the startle response in F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type administered the drug with the percentage of inhibition of the startle response in an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated Prodh gene comprising a DNA sequence of SEQ ID NO:3 which are capable of expressing a Prodh comprising an amino acid sequence of SEQ ID NO:4.
If the percentage of inhibition of the startle response in the medicated mouse is statistically equivalent to the percentage of inhibition in the mouse capable of expressing Prodh comprising a DNA sequence of SEQ ID NO:4, then the drug or agent has the ability to treat schizophrenia or a disease or disorder related thereto.
The PPI test is described infra.
Accordingly it is an object of the invention to provide the DNA sequences of murine and human proline dehydrogenase genes, and the amino acid sequences of murine and human proline dehydrogenase.
It is another object of the present invention to provide heretofore unknown variant alleles of the human PRODH gene, which can be used to map the locus of the human PRODH gene.
It is another object of the invention to provide a heretofore unknown variant allele of the human PODH gene which is a marker for a susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto in a subject, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder in a subject.
It is yet another object of the present invention to provide isolated nucleic acid molecules, optionally detectably labeled, which are hybridizable under standard hybridization conditions to variant alleles of the PRODH gene disclosed herein.
It is yet another object of the present invention to provide variant PRODH proteins, produced from the expression of a variant alleles of the human PRODH gene, or isolated nucleic acid molecules hybridizable to such variant alleles under standard hybridization conditions.
It is yet another object of the present invention to provide antibodies, optionally detectably labeled, having a variant PRODH protein of the present invention as an immunogen, wherein such antibodies may be polyclonal, monoclonal or chimeric.
It is yet another object of the present invention to provide commercial test kits for attending medical professionals to determine the presence of a variant allele of the PRODH gene in a bodily sample taken from a subject. The results of such testing can then be used to determine the subject""s susceptibility to suffer from schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder, or to diagnose such a disease or disorder.
It is yet another object of the present invention to provide a method and associated assay system for screening subjects in order to determine their susceptibility to schizophrenia or a disease or disorder related thereto, and to likewise select an appropriate course of therapy therefor.
It is yet another object of the present invention to provide compositions such as drugs, agents and the like, potentially effective in either potentiating the effects of PRODH, or increasing levels of PRODH in mammalian, especially human patients.
It is still yet another object of the present invention to provide a method for the treatment of mammals to modulate the amount or activity of PRODH or subunits thereof in the mammal, so as to alter the adverse consequences of diminished levels of PRODH, which can result in schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder.
It is a still yet another object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the PRODH, its subunits, their binding partner(s), as well as molecules whose activity or production depends on PRODH; or upon molecules or agents or drugs that control the production, stability and degradation, or that mimic the activities of the PRODH.
It is yet still another object of the present invention to provide methods of determining a susceptibility for, or presence of, schizophrenia, or a disease or disorder related thereto, by determining the levels of an enzyme involved in proline catabolism, wherein such an enzyme comprises PRODH.
It is yet still another object of the present invention to provide numerous methods for the selection of a drug or therapeutic agent to treat potentially schizophrenia, or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP) or major depressive disorder.
It is yet still another object of the invention to provide the DNA sequence of the wild-type murine Prodh gene, and an isolated variant of the allele of the wild-type murine Prodh gene.
It is yet still another object of the invention to provide the amino acid sequence of wild-type murine Prodh protein, as well as a mutant murine Prodh protein.
It is yet still another object of the invention to provide mammalian assays for determining whether a drug or agent has the ability to treat schizophrenia or a disease or disorder related thereto. Such assays involve Pro/Re mice described in the Example, and the PPI test, described below.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.